Renibacterium salmoninarum vaccine and method for its preparation

ABSTRACT

A vaccine and method for treating fish susceptible to infection by Renibacterium salmoninarum is described. The vaccine comprises killed microorganisms that lack intact cell-surface-associated protein p57. The vaccine can be used in combination with additional materials, such as, without limitation, adjuvants, plasticizers, pharmaceutical excipients, antigens other than the cells lacking intact cell-surface-associated protein p57, diluents, carriers, binders, lubricants, glidants, aesthetic compounds, such as flavoring and coloring agents, and combinations thereof. The vaccine may be enteric-coated for oral delivery. The enteric coating generally comprises a polymer coating that is impervious to dissolution and/or degradation in the stomach, but is dissolved upon passing to the higher pH environments of the intestine. A preferred embodiment of the vaccine is made using spherical sugar microspheres. The microsphere is coated with a first layer comprising the killed Renibacterium salmoninarum microorganisms lacking intact cell-surface-associated protein p57. The sugar microsphere is then coated with a second enteric-coating layer comprising a material that is impervious to dissolution and/or degradation in the stomach of the fish.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under USCS-CSRS#92-34123-7665 and USDA (WRAC) #91-38500-6078. The Government may havecertain rights in the invention.

FIELD OF THE INVENTION

This invention concerns a vaccine and method for treating fishsusceptible to infection by Renibacterium salmoninarum.

BACKGROUND OF THE INVENTION

Bacterial kidney disease (BKD) results from infection by Renibacteriumsalmoninarum. BKD is a chronic and systemic disease that generally leadsto mortality in juvenile and adult salmonids, both in fresh water andmarine environments. Bacterial Kidney Disease of Salmonid Fish, Annu.Rev. Microbiol., 35:273-298. Salmonids are fish of the familySalmonidae, which are soft-finned fishes such as salmon, trout, charsand whitefishes.

Renibacterium salmoninarum is a slow-growing gram-positive bacterium.The bacterium is endemic in wild anadromous (migrating up rivers fromthe sea to breed in fresh water) salmonid populations on both coasts ofNorth America, and has been found in wild Atlantic salmon and sea trout.After infection, Renibacterium salmoninarum localizes in the kidney fromwhich infection rapidly becomes systemic.

Farming of marine species is an ancient practice, and aqua culturing offish has increased significantly over the last twenty years. Commercialaqua culturing requires maintaining high densities of cultured fish.This increases the likelihood of economic loss from diseases such as BKDrelative to less-dense fish populations. Although the actual lossesattributed to BKD have not yet been calculated, the disease is known tobe one of the most important bacterial diseases affecting residentanadromous salmonid stocks in the Pacific Northwest. Because BKD is oneof the most prevalent diseases of cultured salmonids, it has had asignificant economic impact on the fishing and aqua culture industries.

There still are limited effective methods for controlling BKD despiteits economic impact. One reason for this is that the bacteria is capableof adjusting to different conditions as an intracellular parasite, andhas the ability to survive and multiply in phagocytic cells (cells thatengulf and digest foreign bodies). Current approaches to managing BKDoutbreaks include stress reduction, quarantine, chemotherapy (antibiotictreatment), total destruction of the infected population and completesterilization of the facilities. These approaches to BKD infection arenot commercially appealing, and are difficult to administer to largefish populations.

There are no known vaccines effective for treating fish susceptible toinfection by BKD, despite the continuous efforts by those skilled in theart. For instance, McCarthy reported an attempt to vaccinate fishsusceptible to BKD using two preparations of formalin-inactivated cellsof Renibacterium salmoninarum. McCarthy et al., Immunization of RainbowTrout, Salmo gairdneri, Against Bacterial Kidney Disease: PreliminaryEfficacy Evaluation, J. of Fish Dis., 7:65-71 (1984). The bacterins wereadministered without adjuvant by IP-injection, immersion, or two-stephyperosmotic infiltration. No significant protection was afforded bythese methods.

Furthermore, Kaattari et al. have treated salmonids with a number ofpotential immunogens in an attempt to confer immunity to fishsusceptible to BKD infection. These immunogens included cell-wallfractions, fractured cells and extracellular products. Kaattari et al.,Development of a Vaccine for Bacterial Kidney Disease, Bonneville PowerAdministration Final Report, (1990). These immunogens were administeredby intraperitoneal injection, orally, and by immersion with and withoutadjuvant. None of these early preparations protected fish. In fact, someof these preparations exacerbated the disease.

The route of delivering vaccines often is an important factor for thesuccessful vaccination of fish. Intraperitoneal vaccination is generallythe most effective method for vaccinating any species, even though IPvaccination is labor intensive. Immersion is another vaccination method,which is widely used on smaller fish (fish that weigh less than about 10to 15 grams). The standard immersion method involves exposing fish tothe vaccine in aerated standing water for a minimum of 20 seconds. Thedisadvantage of immersion vaccination is that it is limited by theweight of fish that can be immunized per unit volume of vaccine. And,immersion vaccination usually provides lower levels of immunity thanother techniques, due to the stress it causes fish.

SUMMARY OF THE INVENTION

Based on the discussion provided above, it is apparent that a vaccine isneeded for protecting salmonids against infection by Renibacteriumsalmoninarum. The present invention provides such a vaccine, as well asa method for treating fish using the vaccine. The vaccine compriseskilled Renibacterium salmoninarum microorganisms that are devoid ofintact cell-surface-associated protein p57. Although vaccines can bemade using virtually any strain of Renibacterium salmoninarum, operativevaccines have been made using Renibacterium salmoninarum microorganismshaving the identifying characteristics of a microorganism selected fromthe group consisting of Renibacterium salmoninarum ATCC strain 33209 andRenibacterium salmoninarum D6 isolate. The vaccine can be used incombination with additional materials, such as, without limitation,materials selected from the group consisting of adjuvants, plasticizers,pharmaceutical excipients, antigens other than cells lacking intactcell-surface-associated protein p57, diluents, carriers, binders,lubricants, glidants, aesthetic compounds, such as flavoring andcoloring agents, and combinations thereof.

The vaccine also may be enteric-coated for oral delivery. The entericcoating protects the vaccine from proteases and from the relatively lowpH levels of the stomach. This allows the vaccine to reach the hindgutassociated with lymphoid tissue, which maximizes the effectiveness ofthe vaccine for protecting fish. The enteric coating typically comprisesa polymer coating that is unaffected by acidic pH, but which isdissolved upon passing to the higher pH environments of the pyloriccaecum and intestine. The pH of salmonid stomachs varies from about 1.5to about 4.8. The physiologic pH rapidly increases in the intestine ofthe fish to pH values of greater than about 5, and continues to increaseto a pH of about 8 in the anus region of the fish. As a result, thepolymer coating should not dissolve until in an environment where the pHis greater than about 5.0 and less than about 8. As a result,enteric-coating materials useful for the invention may be selected fromthe group consisting of enteric-coating materials, particularlypolymeric materials, that dissolve in a liquid having a pH of from about5 to about 8.

Oral administration is a generally preferred method of vaccinating fishagainst BKD using vaccines of the present invention. Oral vaccinationsprovide an ideal method for the mass administration of the vaccine tofish. Oral vaccination also is not limited by the size of the fish thatcan be handled, and it reduces the stress on the fish associated withimmersion and IP vaccination. Furthermore, oral vaccines offer theadditional advantage of stimulating mucosal immunity.

A preferred embodiment of the vaccine is made by coating spherical sugarmicrospheres (beads) with vaccine formulations. The beads can bevirtually any material, now known or hereinafter developed, that isuseful for delivering pharmacological materials. By way of example andwithout limitation, dextrose beads have been shown to be useful forforming such beads. The beads generally have a mesh size of from about10 to about 60 mesh, preferably from about 20 to about 35 mesh, and evenmore preferably from about 25 to about 30 mesh.

The beads generally are coated with a first layer comprising the killedRenibacterium salmoninarum microorganisms lacking intactcell-surface-associated protein p57. This coating also may compriseadditional materials, such as materials selected from the groupconsisting of adjuvants, plasticizers, pharmaceutical excipients,antigens other than cells lacking intact cell-surface-associated proteinp57, diluents, carriers, binders, lubricants, glidants, aestheticcompounds, such as flavoring and coloring agents, and combinationsthereof. For instance, a disintegrant or a super disintegrant often isused to help disperse the material once it is ingested. One example of asuper disintegrant is sodium starch glycolate.

The bead is then coated with a second coating layer comprising anenteric-coating layer. This layer generally is a polymeric layer whereinthe polymer is impervious to dissolution and/or degradation in thestomach of the fish, but does dissolve upon passing out of the stomach.That is, the polymer generally is impervious to dissolution in anaqueous media having a low pH, such as a pH of less than about 5, but isdissolved by an aqueous media having a pH value of from about 5 to about8. There are numerous materials that are potentially useful for coatingthe beads as discussed in detail below. Solely by way of example, apolymeric material currently known to be suitable for coating the beadsis poly(methylacrylic acid-ethyl acrylate).

A preferred embodiment of the second bead coating comprises a mixturethat includes about 2 weight percent to about 50 weight percentpoly(methylacrylic acid-ethyl acrylate), less than about 50 weightpercent of a plasticizer, such as less than about 10 weight percentdibutyl sebacate and less than about 10 weight percent triethyl citrate,and a material that reduces particle agglomeration during the coatingprocess, such as talc. Unless noted otherwise, the weight percentsstated in this application are based on the final dry weight of thecoated beads.

One skilled in the art also will realize that the BKD vaccine of thepresent invention can be used in combination with immunostimulants, suchas β-glucans. The immunostimulant may be incorporated into theformulations coated onto the microspheres so that the immunostimulant isreleased by the beads following the administration thereof to fishsusceptible to infection by Renibacterium salmoninarum. The beads can becoated so that the immunostimulant is released prior to the release ofthe BKD vaccine. This is believed to prime the immune system.Alternatively, the BKD vaccine of the present application may bereleased prior to the immunostimulant. As still another possibility,immunostimulants may be administered by a method other than that chosenfor the delivery of the BKD vaccine. For instance, the BKD vaccine mightbe orally administered and the immunostimulant administered by IPinjection or by immersion, either prior to, simultaneously with, orafter the administration of the BKD vaccine.

The present invention also provides a method for reducing the infectionof fish susceptible to infection by virulent strains of Renibacteriumsalmoninarum. A preferred embodiment of the method comprises firstheating Renibacterium salmoninarum microorganisms to a temperature of atleast about 37° C. to produce Renibacterium salmoninarum microorganismslacking intact cell-surface-associated protein p57. Nonpareil sugarbeads, having a preferred mesh size of from about 25 to about 30, thenare coated with a first layer comprising a mixture of a superdisintegrant and Renibacterium salmoninarum microorganisms lackingintact cell-surface-associated protein p57. The beads are then coatedwith a second layer comprising a pH-sensitive polymeric material that isdissolved by an aqueous media having a pH of about 5.0 or greater,thereby producing an enteric-coated vaccine. The coated beads are orallyadministered to fish in an amount sufficient to reduce the infection byfish susceptible to infection by Renibacterium salmoninarum. The methodmay also comprise the step of treating fish susceptible to infection byRenibacterium salmoninarum with an immunostimulant either before,simultaneously with, or after the step of administering the vaccine tofish.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B is a Western blot (A) and total protein stain (B) ofRenibacterium salmoninarum cells after treatment at 37° C. followed byformalin incubation at 17° C.

FIG. 2 is a schematic representation of an oral enteric-coated vaccineaccording to the present invention.

FIGS. 3A-3D are graphs showing the percent survival of fish over timefollowing challenge with Renibacterium salmoninarum, wherein thechallenged fish had been IP injected with one embodiment of a vaccineaccording to the present invention.

FIG. 4 is a graph of titers of Renibacterium salmoninarum antibodiesfrom chinook salmon immunized with p57⁻ or p57⁺ whole Renibacteriumsalmoninarum cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides the first known vaccine and method foreffectively protecting fish susceptible to infection by Renibacteriumsalmoninarum microorganisms. A detailed discussion follows concerninghow to make the claimed vaccine, as well as how to administer thevaccine to fish. Experimental data also is presented which demonstratesthat the vaccine is effective for protecting fish from infection byRenibacterium salmoninarum.

I. DEFINITIONS

A number of definitions are provided below. These definitions areprovided solely for the convenience of persons reading this disclosure.These definitions are not intended to narrow the scope of such terms todefinitions less encompassing than that understood by persons skilled inthe art.

1. "Killed vaccines" generally refer to microorganisms which have beenheat-treated and thereafter treated with some chemical agent, such asformalin. Renibacterium salmoninarum is heat sensitive. It currently isbelieved that heat treatment alone, such as heating to temperaturesgreater than about 35° C., is sufficient to kill the bacterium. However,solely for the purpose of caution, the heat-treated bacterium alsousually are treated with a chemical agent to produce the killed vaccine.

2. "Protective immunity" is the condition induced by the administrationof a vaccine to a fish wherein the susceptibility of the fish toinfection by a particular pathogen is reduced.

3. "Susceptible fish" are those species of fish of which Renibacteriumsalmoninarum is a pathogen and in which the vaccines of the presentinvention are capable of inducing protective immunity. That is, themicroorganism is capable of causing Bacterial Kidney Disease (BKD) insuch a fish and the fish is capable of being protected from such diseaseby vaccination with the vaccines of the present invention. For thepurposes of the present invention, "susceptible fish" includes allsalmonid fish. Salmonid fish include, but are not limited to, pacificsalmon in general (Oncorhynchus sp.), such as rainbow trout(Oncorhynchus mykiss), chinook salmon (Oncorhynchus tshawytscha), cohosalmon (Oncorhynchus kisutch) sockeye salmon (Oncorhynchus nerca) andatlantic salmon (Salmo salar). Both the chinook and coho salmon appearto be particularly susceptible to infection.

4. "Susceptibility to infection" describes the condition of being a hostfor a particular pathogen and of suffering injury from the diseasecaused by that pathogen. The condition of "susceptibility to infection"encompasses a range of susceptibilities. The degree of susceptibility ofa particular fish to infection by a particular pathogen may bedetermined by calculating the LD₅₀ value for this pathogen. Fish speciesless susceptible to infection by a particular pathogen will have ahigher LD₅₀ for that pathogen than a more susceptible fish species.

5. "p57⁻ " is a short-hand notation which refers to cells ofRenibacterium salmoninarum which lack intact cell-surface-associatedprotein p57.

6. "p57⁺ " is a short-hand notation which refers to cells ofRenibacterium salmoninarum which include cell-surface protein p57.

7. "Adjuvant" as used herein refers to any material that enhances theaction of a drug or antigen.

8. "Pharmaceutical Excipient" refers to any inert substance that iscombined with an active drug or antigen for preparing an agreeable orconvenient dosage form.

II. MATERIALS AND METHODS A. Bacterial Strains

Bacterial Kidney Disease (BKD) is caused by a fastidious, slow growingbacterium, Renibacterium salmoninarum. The bacteria presents itself as afacultative (i.e., the bacteria is capable of an adaptive response tovarious environments) intracellular parasite, which also has the abilityto survive and multiply within the phagocytic cell. Renibacteriumsalmoninarum is a gram-positive, short rod (0.08-1.0×0.3-0.5 μm)bacterium. The bacterium is nonmotile, asporogenous, non-acid fast andencapsulated. The guanine-plus-cytosine (G+C) content of the bacteriaaverages about 53-mole percent.

It is likely that all isolates of Renibacterium salmoninarum can be usedto make vaccines according to the present invention. All strains ofRenibacterium salmoninarum produce cell-surface protein p57. And, allstrains of Renibacterium salmoninarum are significantly geneticallyhomogenous, more so than other bacterium, regardless of where thebacteria are isolated. The genetic homogeneity of the Renibacteriumsalmoninarum is a trait fairly unique to the organism. Persons skilledin the art have tried to develop antibodies useful for distinguishingbetween strains of the bacterium. So far, these efforts have provedfruitless. Thus, "strains" when used in connection with Renibacteriumsalmoninarum simply refers to the location where the bacterium wereisolated, and not to some inherent physiological difference between theisolated microorganisms.

Solely to provide specific guidance as to Renibacterium salmoninarumisolates that have been used to develop vaccines, a first such isolatewas cultured from chinook salmon (Oncorhynchus tsawytscha, Oregon) andhas ATCC strain number 33209. A second isolate, isolate D6, was isolatedfrom coho salmon (Oncorhynchus kisutch, held in salt water in Oregon).The D6 isolate was obtained from C. Banner of Oregon State University.The D6 isolate also is discussed in Wiens et al.'s Monoclonal AntibodyAnalysis of Common Surface Protein(s) of Renibacterium salmoninarum,Fish Pathology, 24:1-7 (1989), which is incorporated herein byreference. All strains used to produce vaccines were stored at -70° C.prior to culture.

The following Example 1 describes a method for culturing Renibacteriumsalmoninarum. This and all subsequent examples should, in no way, beconstrued to limit the scope of the present invention to the particularembodiments described.

EXAMPLE 1

Renibacterium salmoninarum ATCC 33209, or Isolate D6, was grown inone-liter volume portions using a 2.5 liter low form, VWR culture flask.The Renibacterium salmoninarum cultures were grown with intermittentshaking at 17° C. using a KDM-II growth medium prepared according to themethod of Evelyn, except without serum supplementation. This medium isdiscussed in Evelyn et al.'s An Improved Growth Medium for the KidneyDisease Bacterium and Some Notes on Using the Medium, Bull. Int. Epiz.78:511-513 (1977), which is incorporated herein by reference. Thebacteria were grown until an optical density of from about 0.4 to about0.8 was generated at about 525 nm. This required approximately 7 to 8days. Seven one-liter volumes of bacteria from the 2.5-liter low-formVWR culture flasks were combined, and then pelleted by centrifugation at6,000×g for about 30 minutes. The pelleted cells were then resuspendedin 100 ml of cold, phosphate-buffered saline solution (PBS; 0.85% NaCl,10 mM NaPO₄, pH 7.2). The cells were then centrifuged a second time at6,000×g. Thereafter, the cells were placed in microfuge tubes and frozenat -70° C. for storage.

B. Bacterial Extracellular Preparations as Vaccination Control

The vaccine preparations of the present invention, which are describedin detail below, were evaluated relative to a number of controlformulations, including extracellular preparations from Renibacteriumsalmoninarum. An extracellular protein (ECP) preparation for use as acontrol was prepared according to the method of Daly et al.'sAgglutination of Salmonid Spermatozoa by Renibacterium salmoninarum, J.Aquatic Animal Health, 1:163-164 (1989), which is incorporated herein byreference. The following Example 2 describes a method for obtaining theECP.

EXAMPLE 2

2 to 4 grams of wet bacterial cells were washed with 100 ml of sterilephosphate buffer saline and then pelleted by centrifugation at 6,000×gfor 30 minutes. The centrifuged cells were then resuspended in 100 ml ofdistilled and deionized water and placed on ice for about one hour. Thecells were then repelleted by centrifugation at 6,000×g. The supernatantwas removed and cell-surface proteins were precipitated with theaddition of powdered ammonium sulfate. The resulting ECP extract wasdialyzed three times against phosphate buffer saline and filtersterilized by passing the extract through a 0.45 μm filter. The proteinconcentration was then determined by the method of Lowry et al.

C. Preparation of Killed Vaccine Lacking Intact Cell-Surface-AssociatedProtein P57

A major component of the ECP is a protein having a molecular weight ofabout 57 kDa. This protein also is known to be a major cell-surfaceprotein. Without limiting the present invention to one theory ofoperation, it currently is believed that the present vaccine protectsfish where previous attempted vaccines and vaccination methods havefailed because cell surface protein p57 is removed from the bacterialcells before being administered to fish as a vaccine.

Heat treatment is a currently preferred method for removing thecell-surface protein p57. However, the present invention encompasses anymethods now known or hereafter developed for removing p57. Although thetemperature used to remove the protein may vary, a temperature of about37° C. is believed to be the optimal temperature for activating anautologous serine protease, and therefore for cleaving off thecell-surface protein. See Rockey et al., Characterization of aRenibacterium salmoninarum Serine Protease Which Digests a MajorAutologous Extracellular and Cell-Associated Protein, Can. J. Micro.,37:758-763 (1991), which is incorporated herein by reference.

The vaccine of the present invention is administered as a killedvaccine. The present invention encompasses any methods now known orhereafter developed for killing Renibacterium salmoninarum cells for useas a vaccine. However, it has been determined that heating the cells toa temperature of about 37° C. kills the microorganism. Renibacteriumsalmoninarum is a relatively heat-sensitive organism. In fact, it islikely that temperatures of less than about 37° C. can be used to killthe bacterium. However, as the temperature is lowered, the time requiredto kill the organisms increases. If heat treatment alone is used to killthe vaccine, then the temperature likely should not be increased to bemuch above 55° C. Otherwise, the serine protease activity may behindered. As a result, the removal of the p57 cell-surface-associatedprotein would be affected.

In order to insure that the bacteria were killed prior to administeringthe vaccine derived therefrom to fish, the heat-treated cells first alsowere fixed with formalin. The step of formalin fixing likely is asuperfluous step, and it currently is believed that heating the cells isa sufficient method for killing the bacterial cells.

The following Example 3 describes a method for preparing killed p57⁻bacterial cells.

EXAMPLE 3

Frozen harvested cells were prepared as described above in Example 1.The frozen cells were thawed from -70° C. and microfuged at 6,000×g. Thecentrifuged cells were then weighed and resuspended in a sufficientamount of cold, sterile phosphate buffer to obtain a concentration ofabout 200 mg cells/ml. The cells were then heated to a temperature ofabout 37° C., and this temperature was maintained for about 48 hours.After the heating step had been completed, the cells were againmicrofuged at 6,000×g, and then resuspended in a 3% formalin-phosphatebuffered saline solution. The formalin-cell mixture was then cooled to atemperature of about 17° C., which was maintained for about 10 hours.The cells then were repeatedly washed with phosphate buffered salinesolutions and reweighed.

FIG. 1 shows a Western blot (A) and total protein stain (B) ofRenibacterium salmoninarum cells following treatment at 37° C. andformalin incubation at 17° C. as described above in Example 3. TheWestern blot was probed with monoclonal antibody 4D3, which recognizesp57. The lanes represented on the gels are as follows: Molecular weight;Lane 1 shows untreated Renibacterium salmoninarum cells; lanes 2-4illustrate three separate treatments of Renibacterium salmoninarum.Lanes 2-4 on both the Western blot and the total protein stain clearlyindicate the absence of a band corresponding to p57. This demonstratesthat the method described in Example 3 effectively removes p57 to belowdetectable limits, thereby producing p57⁻ Renibacterium salmoninarumcells.

D. Antigen Preparation for Vaccine Comparisons

In order to determine the effectiveness of the killed Renibacteriumsalmoninarum cells for developing an intraperitoneal vaccine, bacterialantigens were emulsified in Freund's incomplete adjuvant (FIA) using aVirtis 23 mixer set at 100 units for four minutes. The putative antigensused for the intraperitoneal vaccination consisted of approximately 500μg of heat-treated Renibacterium salmoninarum cells produced asdescribed above in Example 3, 50 μg of cell surface extract obtained asdescribed above in Example 2, and 50 μg from extracellular proteinextracted from culture supernatants.

E. IP Vaccination and Challenge

Coho salmon were injected with the putative antigen preparations in atotal volume of about 0.1 ml. The fish were injected IP andintramuscularly, with a total of about 0.05 ml at each location, using a22 gauge needle. Booster injections were then given to the fish 45 daysafter the primary injection. The booster injection consisted of aboutone half of the volume of the antigen or control preparations used inthe primary injection, although the total volume of material injectedwas the same in both the primary and booster injections. The fish thenreceived a secondary boost 10 days after the first booster shot wasadministered.

The vaccinated fish were challenged by IP injection. The progress of BKDin fish challenged by natural methods is slow. The onset of the diseasemay take as long as a year or more, which makes laboratory testingimpractical. As a result, the fish were initially challenged by IPinjection to accelerate the onset of the disease. The Renibacteriumsalmoninarum used for the challenge were grown for 7 days in KDM-II andthereafter washed with PBS. The cells were then suspended in sterile PBSto obtain a final optical density of about 0.2 at 525 nm. The fish werethen IP challenged with a challenge dose of about 4.1×10⁶ cfu/ml. Theresults of this challenge experiment are summarized in FIG. 3.

FIG. 3 shows the percent survival of coho salmon immunized with variouspotential immunogenic materials obtained from Renibacteriumsalmoninarum. Three tanks of salmon (40 salmon/tank) were injected withthe various materials. The fish were injected with either salineemulsified in Freund's incomplete adjuvant (see graph 3A), extracellularprotein in Freund's incomplete adjuvant (see graph 3B), virtually purep57 obtained from cell wash and emulsified in Freund's incompleteadjuvant (CW, see graph 3C), and p57⁻ Renibacterium salmoninarum cellsin Freund's incomplete adjuvant. Graphs 3A-3D are illustrated havingerror bars which represent two standard errors about the mean for thethree trials.

The results summarized by FIG. 3 indicate that fish receiving p57⁻ had asignificantly increased mean time to death following challenge with liveRenibacterium salmoninarum. FIG. 3D appears to show that all fish IPtreated with p57⁻ cells die; however, one reason for this is because thefish were challenged with a relatively large concentration of pathogenthat would be expected to cause death, even if the fish weresuccessfully immunized by the vaccine. In other words, the largepathogen concentration used for the challenge was intended to causedeath; otherwise, the time required for the challenged fish to die wouldbe too long for practical laboratory investigation. However, it is clearfrom FIG. 3D that the fish treated by IP immunization had asignificantly enhanced mean time to death following pathogen challenge.

FIG. 4 depicts the units of activity/μl for titers of anti-Renibacteriumsalmoninarum antibodies produced upon immunization of chinook salmon.Two groups of chinook salmon (60 fish per group) were IP immunized usingeither p57⁺ or p57⁻ cells. After 86 days following injection, serumsamples were taken and antibody titers were then determined using theprocedure of Kaattari et al. The fish then received a booster shot onday 100 post initial inoculation. On day 128, serum samples were againtaken and the data is presented in FIG. 4. This data clearly shows thatsalmon demonstrate a 20-fold higher titer to p57⁻ cells (C') than dosera taken from fish injected with p57⁺ cells (A') . More specifically,fish receiving p57⁻ cells had an average titer of about 25,703 units ofantiRenibacterium salmoninarum activity/μl. Fish receiving p57⁺ cellshad an average titer following receipt of the booster shot of about1,276 units of anti-Renibacterium salmoninarum activity/μl. Thus,removing p57 cell-surface protein from the bacterial cells used asimmunogenic material for treating salmonids significantly increased theimmunogenicity of the Renibacterium salmoninarum. Moreover, althoughoral administration of the p57⁻ bacterial cells is a preferred deliverymethod, IP injection also effectively elicits an immune response in fishreceiving the bacterial cells.

F. Preparation of Oral Vaccine

After the results were obtained for IP vaccination and challenge, adecision was made to try and develop an oral vaccine. Although thepresent invention is not limited to administering the vaccine orally,oral administration is a currently preferred delivery method. One reasonfor this is that oral administration apparently stimulates the gutassociated lymphoid tissue (GALT) to a greater extent than does IPinjection.

Microspheres have been used for the oral delivery of the presentvaccine. There are several advantages for using microspheres, including:

(1) The microspheres release the antigen which can then be taken up inthe GALT, which is an important part of the secretory immune system.

(2) Entrapped antigens are protected from being degraded by the acidicenvironment of the stomach.

(3) Plural antigens can be administered at the same time.

(4) Pharmaceutical excipients, such as super disintegrants, can beapplied to the microspheres.

(5) Additional materials, such as adjuvants, may be administered alongwith the specific antigen of interest.

(6) Controlled/sustained-release formulations are possible.

The present invention provides a BKD vaccine that can be administeredorally after the antigenic material is applied to microspheres andsubsequently enteric-coated such that the antigenic materially isencapsulated thereby to produce an enteric-coated antigenic microsphere(ECAM). A schematic diagram of one embodiment of an ECAM is illustratedin FIG. 2 as ECAM 2. The illustrated embodiment of the ECAM 2 comprisesa sugar bead 10 of conventional type, at least a first antigen coating12, and an enteric protective layer 14.

One skilled in the art will realize that a number of sugar beadspotentially are useful for forming ECAMs. However, without limitation, acurrently suitable sugar bead is an NF sugar sphere which can beobtained commercially from Ingredient Technology Corporation, ofPennsauken, N.J. The illustrated sugar beads 10 of FIG. 2 are dextrosebeads.

The size of the bead is an important consideration for selecting anappropriate sugar microsphere. Certain bead sizes have been found to betoo big to pass through the pyloric sphincter of the fish, and thereforemay not effectively confer immunity to fish treated with such beads andan oral vaccine. Presently, it appears that a microsphere having a meshsize of from about 10 to about 60 mesh, preferably from about 20 toabout 35 mesh, and even more preferably from about 25 to about 30 mesh,will perform satisfactorily for forming ECAMs of the present invention.

Sugar beads 10 are coated with a first antigen coating 12. As will beapparent from the preceding discussion, the antigens of choice for thepresent invention are p57⁻ killed bacterial cells. Sugar spheres havebeen coated with this, and other potential immunogenic materials, usinga fluidized bed spray coater. The actual coater used for coating suchmicrospheres was obtained from Labline/PRL, of Melrose Park, Ill. Theantigens were applied to the sugar spheres typically as a gelatinizedsolution, such as about a 4% weight/volume solution.

After the antigen coating 12 has been applied to the sugar bead 10, anenteric-protective layer 14 is then be applied to the sugar bead. Aswith the selection of the sugar bead, one skilled in the art willrealize that a number of enteric-coating agents may be used. The presentinvention is directed to any vaccine comprising p57⁻, including any suchmaterial that has been enteric-coated with any coating material nowknown or hereafter developed.

However, solely by way of example, currently suitable enteric-coatingmaterials are non-toxic polymeric materials which resist dissolution atthe pH of the stomach, but which are dissolved once the material passesfrom the stomach to the pyloric caecum and intestines. Preferably, thepolymeric material is dispersible in an aqueous system without the useof organic solvents. The Environmental Protection Agency does notrecommend using organic solvents for use in spray-coating procedures.Organic solvents also add to the expense of producing the vaccine.

Table 1 below provides a non-exhaustive list of enteric-protectingpolymeric materials currently believed to be useful for forming thevaccines of the present invention.

TABLE 1 ENTERIC COATING POLYMERIC MATERIALS

(1) Cellulose Acetate phthalate (CAP)

(2) Hydroxypropylmethyl Cellulose Phthalate (HPMCP)

(3) Carboxymethylethyl Cellulose (CMEC)

(4) Hydroxypropylmethyl Cellulose Acetate Succinate (HPMC-AS)

(5) Cellulose Acetate Trimellitate (CAT)

(6) Polyvinyl Acetate Phthalate (PAP)

EUDAGRIT BRAND POLYMERS

(7) EUDAGRIT L-30-D and l 100-55

Poly(ethylacrylate, methacrylic acid),

copolymer having a 1:1 ratio of monomers; dissolves at pH=5.5!

(8) EUDAGRIT L 12.5 and L 100

Poly(methacrylic acid,

methylmethacrylate) tend to dissolve at pH of from about 5.8-6.0.

(9) EUDRAGIT E, RL, RS and NE.

Additional information concerning materials useful for forming coatingsfor the present invention can be obtained by consulting (1) Osterwald'sProperties of Film-Formers and Their Use In Aqueous Systems,Pharmaceutical Research, 2:14-18 (1985), and (2) Aqueous PolymericCoatings for Pharmaceutical Dosage Forms, edited by J. W. McGinity,Marcel Publishing (1989). Each of these references is incorporatedherein by reference.

A polymeric material that has been used to form vaccines for the presentinvention is poly(methacrylic acid-ethylacrylate). This material iscommercially available from Rohm Pharma of Weiterstadt, Germany, asEUDRAGIT™ L-30D. The polymeric material was applied in the same manneras the antigen to form enteric-protected sugar spheres.

Persons skilled in the art also will realize that additional materialscan be used in combination with the enteric-coating materials to formthe enteric-coated antigen microspheres. For instance, plasticizersoften are used to form pharmaceutical preparations. Pages 17 and 68 ofAqueous Polymeric Coatings for Pharmaceutical Dosage Forms, supra,provide a list of plasticizers commonly used for pharmaceuticalpreparations. The following Table 2 also provides a non-exhaustive listof useful plasticizers.

TABLE 2 PLASTICIZERS

(1) Polyethylene glycol 200 (PEG 200; 200 refers to the averagemolecular weight)

(2) Polyethylene glycol 400 (PEG 400)

(3) Polyethylene glycol 1000 (PEG 1000)

(4) Polyethylene glycol 4000 (PEG 4000)

(5) Polyethylene glycol 6000 (PEG 6000)

(6) Propylene glycol

(7) PVPK-90

(8) Glycerin or Glycerol

(9) Diethyl Phthalate

(10) Oleic acid

(11) Isopropyl myristate

(12) Liquid paraffin or mineral oil

(13) Triacetin

(14) Glycerol monostearate

(15) Dibutyl Sebacate

(16) Triethyl citrate

(17) Tributyl Citrate

(18) Acetylated monoglyceride

(19) Dibutyl phthalate

(20) Acetyl tributyl citrate

(21) Castor oil

(22) Glycerol tributyrate

Disintegrants, including materials generally considered by those skilledin the art to be super disintegrants, also often are used in combinationwith the enteric-coating material to facilitate the disintegration ofthe microsphere and release of the vaccine. Any disintegrant now knownor hereafter developed likely will work for forming the vaccines of thepresent invention. Solely by way of example, sodium starch glycolate(SSG, Explotab®, Edward Mendell) is a super disintegrant currently knownto be useful for practicing the present invention.

Vaccines produced using spray-coating devices tend to agglomerate whileair-entrained. To alleviate the agglomeration, a "free-flowing" materialmay be added to the coating mixture. A number of "free-flowing"materials potentially are useful for practicing the invention, and theinvention should be interpreted as being broad enough to cover any suchadditives now known or hereafter developed. Solely by way of example,useful materials for preventing the agglomeration of the vaccines duringthe spray coating operation may be selected from the group consisting oftalc, magnesium stearate, silicone, silicon oxide, and combinationsthereof. For the present invention, it has been found that talcefficiently alleviates agglomeration during the coating process, isreadily available, and hence is a currently preferred material foralleviating agglomeration.

Example 4 below describes a method for producing an ECAM according tothe present invention. EXAMPLE 4

100 grams of 25-30 or 30-35 mesh size NU-PARELL® PG, NF Sugar Sphereswere obtained from Ingredient Technology Corporation of Pennsauken, N.J.These sugar spheres were loaded into a Lab-line/PRL fluid-bed bottomspray coater preheated to a temperature of about 60° C. (microspheresalso have been coated at temperatures of from about 37° C. to about 70°C.). The temperature of the sugar spheres was allowed to equilibratewith that of the coating unit. A 22.5 ml aqueous mixture of a vaccineand a suitable binder comprising from about 0.03 weight percent to about4 weight percent BKD vaccine (p57⁻ cells, weight percent based on theweight of the sugar beads to be coated in the coating chamber), andgelatin was prepared. A super disintegrant (either 5%, 9% or 12%) sodiumstarch glycolate (SSG, Explotab®, Edward Mendell, Patterson, N.Y.)) wasadded to this mixture. The 12% SSG released antigen the fastest, andhence is a preferred amount of SSG disintegrant useful for the presentinvention. The sugar spheres were then placed into the preheated coatingapparatus, which was equipped with an 0.8 mm bottom spray nozzle. Theoperating nozzle pressure of the apparatus was about 18± psi, and theblower speed was set at 40 to 50% of full capacity. This caused freemovement of the beads in the coating machine. The BKD vaccine wasconstantly delivered to the nozzle by a peristaltic pump (Gilson MedicalElectronics, Middleton, Wis.) at rate of about 2 to 3 ml/minute.

Once the sugar-sphere coating process was completed, the pellets weredried in the coating chamber for about 5 minutes using the sametemperature and air flow stated above. The antigen-coated beads wereremoved from the coating chamber and kept overnight in an oven that washeated to a temperature of 37° C. This helped remove residual moisturebefore the enteric-coating was applied.

An enteric-coating mixture was then formed comprising about 30% (w/v)Eudragit L-30D (PMA-EA, Eudragit™ L-30D, Rohm Pharma, Weiterstadt,Germany), less than about 10 weight percent dibutyl sebacate (DBS, SigmaChemical Co.), less than about 10 weight percent triethyl citrate (TEC,Aldrich Chemical Company, Inc.) and talc (Matheson Coleman & Bell, EastRutherford, N.J.). This mixture was applied to the antigen-coated sugarbead in the same manner as the antigen was applied, thereby forming anECAM according to the present invention. About 20% (w/w) Eudragit L-30Dwas applied to the beads based on the final dry weight of antigen-loadedbeads.

The following enteric-coating formulations have been applied to sugarbeads to from coated vaccine microspheres.

                  TABLE 3    ______________________________________    ENTERIC-PROTECTED FILM POLYMER FORMULATION FOR    100 G OF 30-35 MESH-SIZE ANTIGEN-COATED VACCINE BEADS    COMPONENTS  GRAMS         WT. IN DISPERSION    ______________________________________    EUDAGRIT L-30D                15 g (Solids) 51.3                (15.4% w/w                based on                antigen loaded                beads; 70%                w/w based on                total polymer                & Plasticizer                solids)    TEC         3.3 g Solids) 3.3    (Triethyl Citrate)                (3.3% w/w                based on                antigen loaded                beads; 15% w/w                based on total                polymer &                Plasticizer                solids)    DBS         3.3 g (Solids)                              3.3    (Dibutyl sebacate)                (3.3% w/w                based on antigen                loaded beads;                15 w/w based                on total polymer                & plasticizer                solids)    Talc        1.1 g         1.1                (5% w/w based on                total polymer                & plasticizer                solids)    Water       N/A           51.3*    ______________________________________     Enteric coating increased weight of microspheres to a final weight of     about 123.1 g.     Total entericcoating solids = 21.3% w/w based on dried antigen loaded     beads.     *Water was added to make the final suspension about 20% w/v.

                  TABLE 4    ______________________________________    ENTERIC-PROTECTED FILM POLYMER FORMULATION FOR    100 G OF 25-30 MESH-SIZE ANTIGEN-COATED VACCINE BEADS    COMPONENTS  GRAMS         WT. IN DISPERSION    ______________________________________    EUDAGRIT L-30D                14 g (Solids) 46.7                (14.0% w/w based                on antigen loaded                beads; 70% based                on total polymer                & Plasticizer                solids)    TEC         3.0 g (Solids)                              3.0    (Triethyl Citrate)                (3.0% w/w based                on antigen loaded                beads; 15% w/w based                on total polymer &                Plasticizer solids)    DBS         3.0 g (Solids)                              3.0    (Dibutyl sebacate)                (3.3% w/w based                on antigen loaded                beads; 15 w/w based                on total polymer &                plasticizer solids)    Talc        1.0 g         1.0                (5% w/w based                on total                polymer &                plasticizer solids)    Water       N/A           46.7*    ______________________________________     Enteric coating increased weight of microspheres to a final weight of     about 121 g.     Total entericcoating solids = 21% w/w based on dried antigen loaded beads

                  TABLE 5    ______________________________________    ENTERIC-PROTECTED FILM POLYMER FORMULATION FOR    100 G OF 20-25 MESH-SIZE ANTIGEN-COATED VACCINE BEADS    COMPONENTS   GRAMS       WT. IN DISPERSION    ______________________________________    EUDAGRIT L-30D                 11.9 g (Solids)                             39.7                 (11.9%                 w/w based                 on antigen                 loaded beads;                 70% based                 on total polymer                 & Plasticizer                 solids)    TEC          2.55 g (Solids)                              2.55    (Triethyl Citrate)                 (3.0%                 w/w based                 on antigen                 loaded beads;                 15% w/w based                 on total polymer                 & Plasticizer                 solids)    DBS          2.55 g (Solids)                              2.55    (Dibutyl sebacate)                 (3.3%                 w/w based                 on antigen                 loaded beads;                 15 w/w based                 on total polymer                 & plasticizer                 solids)    Talc         .85 g        0.85                 (5% w/w based                 on total                 polymer &                 plasticizer                 solids)    Water        N/A         39.7*    ______________________________________     Enteric coating increased weight of microspheres to a final weight of     about 117.9 g.     Total entericcoating solids = 17.9% w/w based on dried antigen loaded     beads.

                  TABLE 6    ______________________________________    ENTERIC-PROTECTED FILM POLYMER FORMULATION FOR    100 G OF 14-18 MESH-SIZE ANTIGEN-COATED VACCINE BEADS    COMPONENTS    GRAMS      WT. IN DISPERSION    ______________________________________    EUDAGRIT L-30D                  7.7 g (Solids)                             25.7                  (7.7%                  w/w based                  on antigen                  loaded beads;                  70% w/w based                  on total                  polymer                  & Plasticizer                  solids)    TEC           1.65 g (Solids)                              1.65    (Triethyl Citrate)                  (1.65%                  w/w based                  on antigen                  loaded beads;                  15% w/w based                  on total                  polymer &                  Plasticizer                  solids)    DBS           1.65 g (Solids)                              1.65    (Dibutyl sebacate)                  (1.65%                  w/w based                  on antigen                  loaded beads;                  15 w/w based                  on total                  polymer &                  plasticizer                  solids)    Talc          .55 g       0.55                  (5% w/w based                  on total                  polymer &                  plasticizer                  solids)    Water         N/A        25.7*    ______________________________________     Enteric coating increased weight of microspheres to a final weight of     about 111.6 g.     Total entericcoating solids = 11.6% w/w based on dried antigen loaded     beads.

Table 7 lists typical parameters that were used to coat the microspheresusing the Labline/PRL spray coater.

                  TABLE 7    ______________________________________    TYPICAL PROCESS CONDITIONS FOR    ANTIGEN OR ENTERIC-FILM COATING    Parameter          Amount or Setting    ______________________________________    Bed load           100 grams    Wurster Insert     bottom spray    Pump               peristaltic    Column             7" Wurster    Nozzle Size        0.8 mm    Inlet Temperature  40 or 65° C.    Atomization Air    15-18 psi    Fluidization air   40-50% of    blower             capacity    Flow Rate          2.3-6.5                       ml/min intermittently*    Spray Time         0.5-1.5 hour    Dry Time           about 15 minutes    Coating Level      very    ______________________________________     *Peristaltic pump was manually turned on or off as necessary to control     clumping of beads during the coating process.

Vaccines also have been made which included adjuvants, such asimmunostimulants. Immunostimulants were added to prime the immune systemof the fish to enhance the immune response that occurs as a result ofadministering the vaccine of the present invention. β-glucans, whichfunction well as adjuvants, are commercially available in molecularweights of from about 150,000 to about 700,000. It currently is believedthat all such β-glucans are useful for forming vaccines according to thepresent invention. Thus, the method for forming ECAMs as described abovecan be modified to include a β-glucan adjuvant. One embodiment of amethod for producing ECAMs utilizing β-glucans involved spray coatingthe sugar beads with p57⁻ cells, a thin coat of AQUACOAT brand coatingagent, over which about 140 mg of β-glucan per 100 grams of beads wasapplied. The beads were then spray-coated with the EUDRAGIT L-30Denteric coating.

One embodiment of a β-glucan-containing vaccine is summarized below inTables 8 and 9. The data provided in Table 8 represents the materialsused in an AQUACOAT layer, and Table 9 provides the materials used toform the enteric-coating layer. The process for coating the beads firstinvolved coating microspheres with vaccine, sodium starch glycolate, andgelatin to produce 160 grams of vaccine-loaded beads. A layer of anAQUACOAT composition was then applied over the first microsphere-coatinglayer. A layer of a β-glucan having a molecular weight of about 415,000was then applied over the AQUACOAT layer. A solution of the β-glucan (10mg/ml) was sprayed on the microspheres until 224 mg of the β-glucan wasapplied to the beads. Finally, the microspheres were coated with theenteric-coating composition as summarized in Table 9.

                  TABLE 8    ______________________________________    AQUACOAT COMPOSITION FOR COATING MICROSPHERES    COMPONENTS   GRAMS       WT. IN DISPERSION    ______________________________________    AQUACOAT ®                 1.6 g (Solids)                             5.33                 (1.0%                 w/w based on                 antigen loaded                 beads; 70% based                 on total polymer                 & Plasticizer                 solids)    TEC          0.24 g (Solids)                             0.24    (Triethyl Citrate)                 (.15% w/w based                 on antigen loaded                 beads; 11.5%                 w/w based on                 total polymer &                 Plasticizer                 solids)    DBS          0.24 g (Solids)                             0.24    (Dibutyl sebacate)                 (.15% w/w based                 on antigen loaded                 beads; 15 w/w                 based on total                 polymer &                 plasticizer                 solids)    ______________________________________

                  TABLE 9    ______________________________________    ENTERIC COATING FOR BEADS FIRST    COATED WITH AQUACOAT    COMPONENTS  GRAMS         WT. IN DISPERSION    ______________________________________    EUDAGRIT L-30D                24.6 g (Solids)                              82.1                (24.6% w/w                based on                antigen loaded                beads; 70%                w/w based on                total polymer                & Plasticizer                solids)    TEC         5.3 g (Solids)                              5.3    (Triethyl Citrate)                (5.3% w/w based                on antigen loaded                beads; 15%                w/w based on                total polymer &                Plasticizer                solids)    DBS         5.3 g (Solids)                              5.3    (Dibutyl sebacate)                (5.3% w/w based                on antigen loaded                beads; 15 w/w                based on total                polymer &                plasticizer                solids)    Talc        1.75 g         1.75                (5% w/w based on                total polymer                & plasticizer                solids)    Water                     82.1*    ______________________________________     Enteric coating increased weight of microspheres to a final weight of     about 198.5 g.     Total entericcoating solids = 21.3% w/w based on dried antigen loaded     beads.     Water was added to make the final suspension about 20% w/v.

The formulations discussed above also have been modified as follows. Thesugar beads first were coated with a mixture comprising the BKD vaccine,gelatin, and SSG solution. Thereafter, a layer of about a 1 w/w percentethyl cellulose (AQUACOAT®) was spray coated onto the sugar beads as asealing coat. A β-glucan layer was applied over the AQUACOAT. Finally,the enteric-coating layer (EUDAGRIT L-30-D) was applied. Theseformulations were designed to release the β-glucan first to prime theimmune system. The BKD vaccine is thereafter released.

However, one skilled in the art will realize that this order could bereversed so that the BKD vaccine was released prior to theimmunostimulant. Moreover, it also would be possible to deliver theβ-glucan be means other than enteric-coated microspheres. For instance,if the fish are to be treated with an ECAM, then the immunostimulant(such as a β-glucan) or other desired material could be delivered byimmersion or IP injection. The immersion or IP injection could be doneeither prior to, simultaneously with or after the ECAM is administeredto susceptible fish.

G. Oral Vaccine Preparation Utilizing ECAMs

Fish were challenged with Renibacterium salmoninarum in order todetermine the effectiveness of oral vaccines made according to thepresent invention. The fish selected for challenge by Renibacteriumsalmoninarum first were fed ECAMs produced according to the proceduredescribed above. The ECAMs were mixed uniformly throughout fish meal. Asufficient amount of distilled water was added to the fish meal to forma mull. The mull was then extruded using a conventional extruder, andthe extruded mixture was cut into pellets. Thus, the ECAMS wereincorporated directly into the food supply fed to the fish.

H. Administering Antigens to Fish

For oral administration, fish received ECAM-incorporated feed on anevery-other-day basis for a total of thirty days.

Control fish received intraperitoneal injections. The fish were injectedwith about 500 μg of the vaccine (in a total volume of about 0.1 ml)anterior to the pelvic fin using a 26-gauge needle. The fish received afirst booster shot after 30 days, and a second booster shot 10 daysafter receiving the first booster shot.

Following treatment by both ECAM and IP injection, the fish were allowedto rest for 20 days, and they were then challenged with Renibacteriumsalmoninarum as described below in Example 5. Each of the fivetreatments was performed in triplicate with a total of 25 fish/tank. Tomonitor the humoral responses and pre-challenge soluble antigen titers,five fish per tank were sacrificed. Sera and kidney samples werecollected prior to the challenge.

EXAMPLE 5

The D-6 isolate strain of Renibacterium salmoninarum was grown asdescribed above, and the contents of 3 one-liter flasks were combined.The fish were then exposed to Renibacterium salmoninarum by bathchallenge as described by Elliot and Pascho (1991), Development of aWaterborne Challenge Procedure for Infecting Salmonids withRenibacterium salmoninarum. Abstract, 14th Annual AFS/FHS Meetings, 32ndWestern Fish Disease Conference, Newport, Oreg., which is incorporatedherein by reference. More specifically, fish were placed in tanks andthe water level was then reduced from a volume of about 125 liters to avolume of about 25 liters. The flow of water to the tanks was thenstopped, and supplemental aeration of the tanks was initiated.Thereafter, viable Renibacterium salmoninarum was added to the tanks inamounts sufficient to give a final Renibacterium salmoninarumconcentration of about 4.2×10⁶ cfu/ml as determined by plate count. Thefish were exposed to the bacteria for about 22 hours in the standingaerated water. Water flow to the tanks was then resumed and the tankswere allowed to fill at a rate of about 2.8 liters/minute. The bacteriawas removed from the tanks through normal effluent flow.

I. ELISA-Based Monitoring of Disease Progress

The progress of fish infection following challenge with Renibacteriumsalmoninarum was accomplished using the monoclonal antibody-based ELISAprotocol, with modifications, as described by Rockey et al., MonoclonalAntibody Analysis of the Renibacterium salmoninarum p57 Protein inSpawning Chinook and Coho Salmon, Journal of Aquatic Animal Health. 3,23-30 (1991), which is incorporated herein by reference. Example 6describes a method for monitoring the progress of fish infection.

EXAMPLE 6

Five fish from triplicate challenge treatments were sacrificed in orderto monitor levels of soluble antigen. Samples were taken frompre-challenged fish. Samples also were taken at 50, 90 and 150 daysfollowing challenge of the fish with Renibacterium salmoninarum. Kidneysamples were obtained from each fish and were stored in microfuge tubesheld on ice. The kidney samples were then mixed with cold 1% bovineserum albumin in Tween 20 tris buffered saline (1:1 weight-volume; trisbase, EDTA, NaCl and Tween 20). The samples were homogenized byrepeatedly passing them through a 1 ml syringe. Supernatants werecollected as described by Rockey et al. ELISA's were then performed onall samples according to the protocol of Rockey et al. Incubation timesalso were as described by Rockey et al. Optical densities were measuredat 405 nm using a Titertek Multiscan Plus plate reader that waspurchased from Flow Laboratories. A standard p57 curve was run on everyplate. The concentration of p57 in each sample was calculated asdescribed by Rockey et al. using optical-density values generated fromthe standard curve. It was determined that the assay had a baselinedetection limit of about 1.65 ng/ml. Fish were considered to be infectedwith Renibacterium salmoninarum if the detected level of antigen was atleast 3 ng/ml or greater.

The data obtained from these ELISA evaluations was statisticallyanalyzed to determine if there were any significant differences betweencontrols and fish treated with vaccines according to the presentinvention. These results are presented below in Tables 10 and 11. Thereappeared to be considerable variance between p57 levels in thechallenged fish; therefore, all data was log transformed. The resultsshown below indicate that there was no statistically importantdifference between control studies and vaccinated fish prior to about 90days. However, at ninety days the mean p57 levels (ng/ml) for thevaccinated fish was about 20 ng/ml, whereas the control had mean proteinlevels of about 351 ng/ml. At 150 days the statistical analysis clearlydemonstrates that the fish treated orally with p57⁻ cells had asignificant decrease in the levels of p57, thereby demonstrating theefficacy of vaccines produced according to the present invention.

Table 11 shows the results of serum antibody titers, expressed inactivity/μl, throughout the testing period. These results demonstratethat the serum activity levels for p57⁻ orally treated fish were muchlower than for control fish, or for fish treated by other methods.Specifically, p57⁻ orally treated fish had an activity of about 126units/μl, whereas the mean value for the control fish was about 2060units/μl.

                                      TABLE 10    __________________________________________________________________________    Values expressed are the means of p57 detected (ng/ml) for each    particular treatment at    each sampling date. Standard errors are in parentheses. Asterisk denotes    significant    difference from control p < 0.01, ** = p < 0.03 versus control.                 Mean pre-                       mean 50 days                              mean 90 days                                     mean 150 days    Treatment          number of fish                 challenge                       post challenge                              post challenge                                     post challenge    __________________________________________________________________________    .sup.a Control          15/sample day                 .sup. <3 ng/ml.sup.#                       2.4 (.34)                              351 (352)                                     2070 (1600)    .sup.b Oral p57-          15/sample day                 <3 ng/ml                       1.2 (.2)                              20 (18)                                      1.9 (.419)*    .sup.c NPP p57-          15/sample day                 <3 ng/ml                       1.9 (.51)                              21 (17)                                      2.9 (.59)**    .sup.d Oral p57+          15/sample day                 <3 ng/ml                       1.3 (.3)                              8701 (8600)                                     8403 (5603)    .sup.e ip p57-          15/sample day                 <3 ng/ml                       2.14 (.94)                              12900 (6400)                                     220 (173)    __________________________________________________________________________     .sup.a = control nonantigen coated beads.     .sup.b = ECAM delivered p57- whole cells.     .sup.c = NonpH protected p57- whole cells.     .sup.d = p57+ whole cells.     .sup.e = intraperitoneal injected p57- whole cells.     .sup.#  = below detection limit of assay.

                                      TABLE 11    __________________________________________________________________________    Values expressed are the means of serum antibody units of activity/μl    serum detected for    each particular treatment at each sampling date. Standard errors are in    parentheses.                 Mean pre-                       mean 50 days                              mean 90 days                                      mean 150 days    Treatment          number of fish                 challenge                       post challenge                              post challenge                                      post challenge    __________________________________________________________________________    .sup.a Control          15/sample day    ND-.sup.#    ND-                       2060 (226)                                      2060 (2276)    .sup.b Oral p57-          15/sample day    ND-    ND-    ND-   126 (436)    .sup.c NPP p57-          15/sample day    ND-    ND-   938 (2241)                 1827 (3146)    .sup.d Oral p57+          10/sample day    ND-    ND-   500 (1881)                 3423 (5852)    .sup.e ip p57-          15/sample day                 5800 (1500)                       42400 (52030)                              82000 (210000)                                      14776 (22065)    __________________________________________________________________________     .sup.a = control nonantigen coated beads.     .sup.b = ECAM delivered p57- whole cells.     .sup.c = NonpH protected p57- whole cells.     .sup.d = p57+ whole cells.     .sup.e = intraperitoneal injected p57- whole cells.     .sup.#  = below detection limit of assay.

J. Determination of Antibody Activity

Antibody activity titers were ascertained by the use of an enzyme-linkedimmunosorbent assay (ELISA) as previously described by Arkoosh andKaattari (1990) Quantitation of Fish Antibody to a Specific Antigen byan Enzyme Linked Immunosorbent Assay (ELISA), Techniques in FishImmunology, pp. 15-24, which is incorporated herein by reference. Eachantiserum was titrated on an ELISA plate that was obtained from CostarE.I.A./R.I.A., Certified Surface Chemistry of Cambridge, Mass.Formalin-fixed Renibacterium salmoninarum was used as a coating agent ata concentration of approximately 150 μg/ml. Each plate contained atitration of an antiRenibacterium salmoninarum hyperimmune-serum.

Generally, the detection of serum antibodies is considered a measure ofimmunity. However, the results of the serum antibody titers seem toindicate that serum antibodies are not necessarily an indication ofimmunity in the vaccinated fish. Fish receiving oral vaccines survivedRenibacterium salmoninarum challenge, but typically had lower serumantibody levels than fish receiving an IP injection. Fish receiving IPinjections did exhibit an increased mean-time-to death. All treatmentgroups, other than the ECAM-delivered p57⁻ whole cells and the orallyadministered, non-pH protected p57⁻ whole cells, had significantlyhigher occurrences of p57 in the kidneys of fish challenged withRenibacterium salmoninarum.

Without limiting the present invention to one theory of operation, itappears that the best vaccination results are obtained by inducingmucosal immunity. As a result, serum antibody levels are of lessimportance than mucosal antibody levels. Alternatively, it may be thatthe protective response in the fish is not mediated by antibodies.

The present invention has been described with reference to severalpreferred embodiments. Other embodiments of the invention will beapparent to those skilled in the art from a consideration of thisspecification or practice of the invention disclosed herein. It isintended that the specification and examples contained herein beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

We claim:
 1. A vaccine for treating fish susceptible to infection byRenibacterium salmoninarum comprising killed Renibacterium salmoninarummicroorganisms lacking substantially all intact cell-surface-associatedprotein p57.
 2. The vaccine according to claim 1 and further including amaterial selected from the group consisting of adjuvants, plasticizers,pharmaceutical excipients, antigens other than cells lackingsubstantially all intact cell-surface-associated protein p57, diluents,carriers, binders, lubricants, glidants and aesthetic compounds, andcombinations thereof.
 3. The vaccine according to claim 1 wherein theRenibacterium salmoninarum microorganism is Renibacterium salmoninarumATCC strain
 33209. 4. The vaccine according to claim 1 comprising anenteric coating.
 5. The vaccine according to claim 4 wherein the entericcoating is impervious to dissolution in a stomach of the fish.
 6. Thevaccine according to claim 4 wherein the enteric coating is selectedfrom the group consisting of polymeric enteric coating materials thatdissolve in a liquid having a pH of about 5 or greater.
 7. The vaccineaccording to claim 4 wherein the enteric-coating material is a polymericenteric coating material that dissolves in a liquid having a pH of about5 or greater, and less than about
 8. 8. The vaccine of claim 1, whereinthe vaccine is prepared by heating the Renibacterium salmoninarummicroorganism at a sufficient temperature for a sufficient period oftime to provide a killed microorganism that substantially lacks intactcell-surface associated protein p57.
 9. The vaccine of claim 8, whereinthe microorganism is heated at a temperature of 37°-55° C. for asufficient period of time to kill the microorganism and substantiallyremove intact cell surface associated protein p57.
 10. The vaccine ofclaim 9, wherein the microorganism is encapsulated by an enteric coatingthat dissolves to release the killed microorganism in a pH greater than5.
 11. The vaccine of claim 9, wherein the microorganism is encapsulatedby an enteric coating that dissolves to release the killed microorganismin a pH of 5-8.
 12. An oral vaccine for treating fish susceptible toinfections by Renibacterium salmoninarum comprising:killed Renibacteriumsalmoninarum microorganisms lacking substantially all intactcell-surface-associated protein p57; and an enteric coating thatprotects the Renibacterium salmoninarum microorganisms from degradationin a stomach of the fish.
 13. The vaccine according to claim 12 whereinthe microorganism is Renibacterium salmoninarum ATCC strain
 33209. 14.An oral vaccine for treating fish susceptible to infection byRenibacterium salmoninarum, comprising:microspheres having a mesh sizeof from about 10 to about 60 mesh; a first microsphere coating layercomprising killed Renibacterium salmoninarum microorganisms lackingsubstantially intact cell-surface-associated protein p57; and at least asecond microsphere coating layer comprising an enteric coating layerthat is impervious to dissolution in a stomach of the fish.
 15. Thevaccine according to claim 14 and further including a material selectedfrom the group consisting of adjuvants, plasticizers, pharmaceuticalexcipients, antigens other than cells lacking intactcell-surface-associated protein p57, diluents, carriers, binders,lubricants, glidants, and aesthetic compounds, and combinations thereof.16. The vaccine according to claim 15 wherein the adjuvant is adisintegrant or a super disintegrant.
 17. The vaccine according to claim15 wherein the pharmaceutical excipient is a β-glucan.
 18. The vaccineaccording to claim 15 wherein the enteric-coating layer comprises apolymeric organic material that dissolves in a liquid having a pH ofabout 5 or greater.
 19. The vaccine according to claim 18 wherein theenteric-coating layer comprises poly(methylacrylic acid-ethyl acrylate).20. The vaccine according to claim 15 wherein the enteric coating layercomprises from about 2 weight percent to about 50 weight percentpoly(methylacrylic acid-ethyl acrylate), less than about 10 weightpercent dibutyl sebacate, less than about 10 weight percent triethylcitrate, and talc.
 21. An oral vaccine for treating fish susceptible toinfection by Renibacterium salmoninarum, comprising:microspheres havinga mesh size of from about 25 mesh to about 30 mesh; a coating layercomprising killed Renibacterium salmoninarum microorganisms lackingsubstantially all intact cell-surface-associated protein p57; anenteric-coating layer comprising a polymeric organic material that isimpervious to dissolution in a stomach of the fish; and a materialselected from the group consisting of adjuvants, plasticizers,pharmaceutical excipients, antigens other than cells lackingsubstantially all intact cell-surface-associated protein p57, diluents,carriers, binder, lubricants, glidants, aesthetic compounds, andcombinations thereof.
 22. A vaccine for treating fish susceptible toinfection by Renibacterium salmoninarum, comprising Renibacteriumsalmoninarum heated to a temperature of about 37° C. for about 48 hours.